Biological sampling

1. Biological sampling DREAMS-21/22

2. Atmospheric bacteria • Recently, scientists have begun to analyze the biological composition of the upper troposphere and lower stratosphere1 • Bacteria, fungal spores, and other microscopic biogenic materials could play an integral role in climate, cloud formation2, and other weather phenomena • Certain scientists have observed possible bacterial adaptations to the high-UV, low pressure, and low nutrient environment of the upper atmosphere3 • …leading them to speculate whether or not these adaptations allow the bacteria to survive independently, forming a novel ecosystem, or “microbiome”

3. Possible applications • In 2012, students at Whitecastle University cultured stratospheric bacteria B. stratophericusto create a biofilm that generates electricity5 • A NASA scientist has noted that the adaptations of stratospheric bacteria could be used as a model for terraforming new planets4 • Human health implications: the majority of microscopic matter in the atmosphere comes from desert dust, and desertification and development are contributing to this load. Atmospheric pathogenic microbes have the potential to cause numerous health issues6

4. Collecting Samples: on the ground • Remove agar plates from refrigerator. • Allow plates to warm to room temperature for about an hour before taking the sample. • Swab area of interest with sterile swab or inoculating loop. • Immediately transfer to agar plate by sweeping the loop/swab across the surface of the agar. Streak back and forth to distribute bacteria evenly. • Replace cover on dish, tape closed, and label each dish with the bacteria source. • Place upside down in incubator, at 90 F. Incubate for 24-48 hours, checking daily for growth.

5. Collecting samples: in Flight • Wearing sterile gloves, prepare 9 nutrient agar plates according to directions included with agar. • With a permanent marker, label the cover and bottom of each agar plate. Label three “flight”, three “control”, and three “site”. Number each plate in a set 1-3. Seal them tightly with tape. Leave the control plates in the lab in a refrigerator. • Bring the six plates labeled “site” and “flight” in an ice-filled cooler to the launch site. An hour before launch, mount to the top the boxes with Velcro the 3 plates labeled “flight”. • At some point, remove plate labeled “site 1” from the cooler, and let sit for one hour. Unseal and open for 30 minutes, near the “flight” plates, for 30 minutes. Minimize the amount of contact with team members. • At the last possible moment before launch, unseal and open the plates labeled "flight". • Immediately after the balloon lands on the ground, seal the flight plates. • For the first balloon landing, place the precooled “site 2” plate near the landing point and let sit for several minutes. Do the same with the “site 3” plate at the second balloon landing. • Place all plates back in cooler or storage. • Incubate all plates at 37°C overnight. Check the every day for growth. • Incubate for up to 48 hours.

6. Viewing samples • After visible colonies develop, dip a sterile inoculating loop into a colony, and spread it across a microscopy slide—adding a drop of clean water as needed—to form a thin film over the slide. The bacteria needs to be relatively uniformly distributed over the slide. • Run the slide over a heat source to heat fix the bacteria on the slide. If using a Bunsen burner, hold the slide about several inches over the flame so that the slide is warm to the touch. Repeat this three times. • Add iodine crystal violet dropwiseso that the bacteria on the slide is completely covered. Let stand for 30 seconds, then rinse with water. • Wash the stain off with water. Flood the slide with gram iodide, and let stand for 30 seconds. Rinse. Add ethanol. Let stand for 5 seconds before rinsing with water. • Flood the slide with saffranin. Let stand for about thirty seconds. Rinse with ethanol. • Blot dry with bibulous paper (if the paper is available). • Place under microscope to identify whether or not the bacteria are gram positive or negative. Focus at 10x magnification, view details at 40x magnification. For more acute detail, obtain mineral oil, place a drop on the slide underneath the lens, and change the lens from 40x to 100x so that the lens is immersed. • Perform further biochemical tests based on identity, gram + or -, of the bacteria.

7. Gram + are violet Gram – are red Gram Stain Procedure Video www.youtube.com/watch?v=4LVVJqD7LjM

8. References • 1: Microbiomeof the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications, PNAS. DeLeon-Rodriguez et al, 2013www.pnas.org/content/110/7/2575.full.pdf+html • 2: Ubiquity of Biological Ice Nucleators in Snowfall. Christner et. al. Science, 2008. www.sciencemag.org/content/319/5867/1214.full • 3: How do microorganisms reach the stratosphere ?. International Journal of Astrobiology. Wainwright et. al, 2006 eprints.whiterose.ac.uk/1556/1/wainrightm1.pdf • 4: • 5: Stratospheric superbugs offer new source of power. ScienceDaily, Feb 2012. www.sciencedaily.com/releases/2012/02/120221212614.htm • 6:Atmospheric Movement of Microorganisms in Clouds of Desert Dust and Implications for Human Health. Griffin, Clinical Microbiology Reviews. 2007. cmr.asm.org/content/20/3/459.full.pdf+html